Fluorescence in situ hybridization (FISH) using non-commercial probes in the diagnosis of clinically suspected microdeletion syndromes.

Background & objectives: Microdeletion syndromes are characterized by small (<5 Mb) chromosomal deletions in which one or more genes are involved. These are frequently associated with multiple congenital anomalies. The phenotype is the result of haploinsufficiency of genes in the critical interval. Fluorescence in situ hybridization (FISH) technique is commonly used for precise genetic diagnosis of microdeletion syndromes. This study was conducted to assess the role of FISH in the diagnosis of suspected microdeletion syndrome. Methods: FISH was carried out on 301 clinically suspected microdeletion syndrome cases for the confirmation of clinical diagnosis using non-commercial probes. Of these, 177 cases were referred for 22q11.2 microdeletion, 42 cases were referred for William syndrome, 38 cases were referred for Prader Willi/Angelman and 44 cases were referred for other suspected microdeletion syndromes. Results: FISH was confirmatory in 23 cases only (7.6%). There were 17 cases of 22q11.2 microdeletion, four cases of Prader Willi syndrome and two cases of William syndrome. Interpretation & conclusion: We conclude that FISH should not be the method of choice for clinically suspected microdeletion syndromes. We propose to follow strict clinical criteria for FISH testing or preferably to follow better methods (genotype first approach). Whole genome screening may be used as first line of test and FISH may be used for confirmation of screening result, screening of family members and prenatal diagnosis.

Rapid detection of chromosome X, Y, 13, 18, and 21 aneuploidies by primed in situ labeling/synthesis technique.

AIMS AND OBJECTIVE: Primed in situ labeling/synthesis (PRINS) technique is an alternative to fluorescent in situ hybridization for chromosome analysis. This study was designed to evaluate the application of PRINS for rapid diagnosis of common chromosomal aneuploidy. MATERIALS AND METHODS: We have carried out PRINS using centromere specific oligonucleotide primers for chromosome X, Y, 13, 18 and 21 on lymphocyte metaphase and interphase cells spread. Specific primer was annealed in situ, followed by elongation of primer by Taq DNA polymerase in presence of labeled nucleotides. Finally, reaction was stopped and visualized directly under fluorescent microscope. RESULTS: Discrete centromere specific signals were observed with each primer. CONCLUSION: PRINS seems to be a rapid and reliable method to detect common chromosome aneuploidy in peripheral blood lymphocyte metaphase and interphase cells.

Effect of gonadotropins on chromosome aneuploidy, chromosome mosaicism & sex ratio in mouse preimplantation embryos.

Background & objectives: There are potential risks of major birth defect in IVF (in vitro fertilization) pregnancy as well as IVF-ICSI (intra cytoplasmic sperm injection) pregnancies in comparison with naturally conceived human pregnancies. This increase risk could be due to either gonadotropins used for ovarian stimulation or in vitro culture conditions or multiple pregnancy or combinations of all the factors. The effects of gonadotropins on chromosome aneuploidy, chromosome mosaicism and sex ratio on mouse preimplantation embryos were evaluated through the use of fl uorescence in situ hybridization (FISH). Methods: The study material consisted of 111 preimplantation mouse embryos (2-16 cell stage) in control group and 405 preimplantation mouse embryos in gonadotropin stimulated group from genetically identical Swiss Albino young (6-8 wk) mouse kept in a similar environmental conditions. The study was designed to investigate effect of gonadotropins on chromosome aneuploidy, chromosome mosaicism and sex ratio through the use of FISH technique using chromosome X, Y and 19 probes. All blastomeres of embryos in both groups were assessed. Results: Interpretable FISH results were obtained in 66 embryos in control group and 128 embryos in gonadotropin stimulated group. There was no excess of chromosome aneuploidy (only one case of sex chromosome trisomy in study group; 19, 19, X, Y, Y) or chromosome mosaicism or deviations in sex ratio between the two groups. However, deviation (1.36 M: 1 F in control group & 1.25 M : 1 F in study group) was seen from expected sex ratio (1 M : 1 F) i.e., skewed sex ratio in both the groups. Interpretation & conclusions: Our results showed that gonadotropins used for ovarian stimulation had no effects in causing increase in chromosome X, Y, 19 aneuploidy and mosaicism and skewing of sex ratio in mouse model. A large scale study with more FISH probes on a larger sample size need to be done to confi rm the findings.

A preliminary study on chromosome aneuploidy & mosaicism in early pre-implantation human embryo by fluorescence in situ hybridization.

BACKGROUND & OBJECTIVE: Chromosome aneuploidy plays an important role in infertility, early pregnancy wastage and perinatal mortality. Cytogenetic & fluorescent in situ hybridization (FISH) studies on developmentally arrested and morphologically poor embryo have shown high frequency of chromosomal abnormality and mosaicism. In this study, we attempted to evaluate chromosome aneuploidy and mosaicism on human embryos through the use of FISH. METHODS: Sixty one grade IV un-transferable embryos were obtained from 25 patients undergoing in vitro fertilization (IVF). Forty six embryos were studied by FISH; 15 were lost during transport and handling. FISH probes (non-commercial) for centromeres of chromosome X, Y, 1 and 18 were used for the study. Zona of embryos were dissolved in 0.01N HCl containing 0.1 per cent Tween 20 for 2-3 min. RESULTS: Interpretable FISH results were obtained in 24 embryos. Nineteen embryos (79.2%) were disomic (normal) for chromosome X/Y or 1/18 and five (20.8%) were abnormal. Among five abnormal embryos two were triploidy (from same patient), one was double mosaic aneuploidy, one was mosaic aneuploidy and one was trisomy for sex chromosome (XXY). There was eleven embryos with presence of Y chromosome i.e., male and three embryos were female. INTERPRETATION & CONCLUSION: Skewing of sex ratio (11M vs. 3F) and low chromosome aneuploidy were observed in this preliminary study, however, it will be premature to conclude as the numbers of embryos studied were limited and so were the numbers of FISH probes used.

Skewed sex ratio and low aneuploidy in recurrent early missed abortion.

BACKGROUND AND OBJECTIVES: Conventional cytogenetic studies have revealed more number of females in spontaneous abortion and it has been assumed that a large proportion of those were resulted from maternal contamination and overgrowth of maternal decidua in long term culture. In this study we have attempted to overcome difficulties of conventional cytogenetics by using meticulous tissue dissection and molecular methods onto uncultured chorionic villous tissue thus bypassing long term culture to find out true sex ratio and frequency as well as type of common aneuploidy in early missed abortions. METHODS: Early missed abortion products (n=58) were collected from recurrent aborter in and around Lucknow, India, over a period of three years. All the cases were selected on the basis of ultrasonography diagnosis. Chorionic villous tissue was cleaned from maternal tissue and processed for conventional as well as molecular cytogenetic analysis. RESULTS: Conventional cytogenetics was successful in 15, of which 12 were females and 3 males. There were 3 cases of chromosomal abnormality, including one false. Interphase FISH with X, Y, 1, 9, 12, 16, 18 and 13/21 probes was carried out in all 58 cases. There were 43 females and 15 males. Four cases of chromosomal abnormality were detected by interphase FISH (6.9%). Comparative genomic hybridization was successful in 8 cases (6 females and 2 males). There was no aneuploidy; however, suspected gain and losses were seen in 4 cases. INTERPRETATION AND CONCLUSION: Our results suggested skewing of sex ratio (M : F, 1 : 2.9 ) and low aneuploidy rate, indicating that in early missed abortion from recurrent spontaneous abortion female outnumbers male. The various possibilities with literature support are presented that may serve as a template for future work.

Detection of 22 q11.2 hemizygous deletion by interphase FISH in a patient with features of CATCH 22 syndrome.

We report an eight years female child with clinical and molecular cytogenetic findings consistent with CATCH 22 syndrome characterized by cardiac defect, typical facial dysmorphism, mental deficiency and chromosome 22 q11.2 deletion. Interphase FISH with 22q 11.2 probe demonstrated hemizygous deletion in 98.5% nuclei. Interphase FISH for diagnosis of CATCH 22 syndrome has not been reported previously from India to our knowledge.

A preliminary investigation of genomic screening in cervical carcinoma by comparative genomic hybridization.

BACKGROUND & OBJECTIVE: Available clinical, radiological and histopathologic risk factors are not adequate for accurate prognosis in uterine cervix carcinoma. Hence there is a need to identify indicators to select high risk cases. Most cancers occur and progress through step-wise somatic genetic mutations. Thus screening of whole genome for specific genomic alterations and its outcome following treatment may predict prognosis. The present study was carried out to investigate genomic alterations associated with cervical carcinoma and any association of genomic alterations with clinico-pathologic parameters. METHODS: Cervical carcinoma cases (n = 4) were subjected to protocol based clinical evaluation, treatment and follow up as a double blind procedure. Tumour samples were collected before radiotherapy and 3 months after completion of radiotherapy. All the samples were stored at -80 degrees C. Comparative genomic hybridization (CGH) was carried out to screen genomic alterations in all tumour samples obtained before treatment. Conventional fluorescent in situ hybridization (FISH) was carried out to confirm the CGH findings and to follow up post-treatment samples. Patients were followed up for a minimum of one year or until death. RESULTS: The CGH analysis identified genomic losses and gains. The gains were observed mainly in chromosomes 1q 25.1, 3q 26.1, 6q 13-16, 9p 22 and X, and losses in chromosome 10 and 11q21-24. CGH and FISH results were complementary to each other. Of the four patients, two were alive and two were dead at the end of follow up. INTERPRETATION & CONCLUSION: Initial results indicated that persistence of genomic alterations and appearance of giant nucleus was associated with poor prognosis and the same may be used to follow up patient. Similar studies on large sample with longer period of follow up are warranted to validate our result.

Placental chimerism in early human pregnancy.

Background0 : Human chimerism is rare and usually uncovered through investigations of ambiguous genitalia or blood grouping or prenatal diagnosis. Most of the publications on placental chimerism are mainly case reports. There is no systematic search with sensitive techniques for placental chimerism in human. Aim0 : This study was aimed to asses placental chimerism through two sensitive molecular techniques i.e., interphase fluorescent in situ hybridization and quantitative fluorescent PCR. Material and methods0 : Placental chimerism was analyzed using X & Y dual color fluorescent in-situ hybridization onto 154 placentae from natural conceptions, obtained at termination of pregnancy between 7 to 16 weeks of gestation. Results0 : Three cases of placental sex chromosome chimerism were observed (1.95%). Exclusion of maternal contamination and diagnosis was confirmed later by quantitative fluorescent PCR. Conclusion0 : This finding indicates that placental chimerism in early human pregnancy is not rare.

Fowler-like syndrome with extreme oligohydramnios, growth restriction and without muscular hypoplasia.

We report the seventh family of Fowler like syndrome (proliferative vasculopathy and hydrocephaly-hydrencephaly syndrome) and first case from Indian subcontinent. A 35 weeks extremely growth retarded male baby showed enlarged ventricles, thinned out cerebral cortex, diffuse intra-cerebral as well as peri-ventricular calcification, cerebral and corneal vasculopathy, unilateral micro-ophthalmia along with corneal opacity and depressed pulsatile anterior fontanel. This case was different from others concerning association with extreme oligohydramnios (in contrast to polyhydramnios), extreme growth restriction (in contrast to normal growth) and absence of gross muscle hypoplasia. No causative factors like TORCH infection, chromosomal abnormality or positive family history was noted in this case.

Low level of mosaicism in atypical Prader Willi syndrome: detection using fluorescent in situ hybridization.

Prader Willi syndrome (PWS) most commonly is due to paternal micro-deletion of 15q11-q13. Although PWS is not a rare condition, mosaic micro-deletion cases are reported rarely. FISH using PWS micro-deletion probe is the most useful method to detect deletion including mosaicism. In this report we describe a female child with clinical features of atypical PWS and FISH analysis showing mosaicism for deletion in the PWS critical region. This is first mosaic deletion case of PWS from Indian subcontinent.